Background/Aims: The cell of origin for Barrett's epithelium is unknown. By scanning and transmission electron microscopy, we have identified cells with features of both squamous and columnar epithelium at the squamo-Barrett's junction. Recently, by light microscopy we described a multilayered epithelium within Barrett's epithelium that is reminiscent of immature squamous metaplasia of the cervix. To investigate the hypothesis that this multilayered epithelium may be a transitional stage between squamous and Barrett's epithelium, we used cytokeratin immunocytochemistry to examine normal squamous, Barrett's, and the multilayered epithelium. Methods: We investigated 17 endoscopic biopsies taken from the squamo-Barrett's junction of 8 patients with Barrett's epithelium and 3 biopsies from the gastroesophageal junction of 3 patients without Barrett's epithelium. Antibodies to cytokeratins 4 and 13 were used as markers for squamous differentiation and to cytokeratins 8 and 19 as markers for glandular differentiation. Coded samples were evaluated both by immunocytochemical methods using the antibodies cited above and by light microscopy using hematoxylin and eosin staining. Light microscopic features and immunocytochemical staining results were then correlated. Results: In patients with Barrett's epithelium and control patients without Barrett's epithelium, staining with antibodies to cytokeratins 8 and 19 was confined to either the Barrett's or gastric columnar epithelium. Staining with antibodies to cytokeratins 4 and 13 was confined to the adjacent squamous epithelium. In marked contrast, focal areas of multilayered epithelium amidst Barrett's epithelium stained with cytokeratin antibodies for both squamous and columnar epithelium. Conclusions: A focal multilayered epithelium within Barrett's esophagus is described that expresses concurrently both squamous and glandular cytokeratin markers. These findings suggest a multipotential cell as the cell of origin of Barrett's esophagus. By analogy with other metaplastic epithelia, a likely candidate for this multipotential cell is the basal cell of squamous epithelium.